Technical Field
The present invention relates to a novel strain of Trichoderma which can produce cellulase and a method for producing the cellulase therefrom.
Background Art
Cellulase exists widely in various organisms in the nature. It could be produced from bacteria, fungi and animal body. Generally, cellulase used for industrial production is prepared from fungi, typically Trichoderma (Trichodema), Aspergillus (Aspergillus), and Penicillium (Penicillium) fermentation. The cellulase is applied in a food industry and an environmental industry extensively.
Filamentous fungi of phylum (division) Ascomycota, including various Penicillium, Phanerochaete, Agaricus, Neurospora, Humicola, Fusarium, Chaetomium, Magnaporthe, Aspergillus and Trichoderma species, have a key role in degradation of most abundant polymers found in nature. Trichoderma reesei (the asexual anamorph of Hypocrea jecorina) is an important industrial source of cellulase and hemicellulase enzymes. The term cellulase (or cellulase enzymes) broadly refers to enzymes that catalyze the hydrolysis of beta-1,4-glucosidic bonds joining individual glucose units in cellulose polymers. Its catalytic mechanism involves synergistic actions of endoglucanases (E.C. 3.2.1.4), cellobiohydrolases (E.C. 3.2.1.91) and beta-glucosidase (E.C. 3.2.1.21). The term hemicellulase broadly refers to enzymes that catalyze the hydrolysis of various glycosidic bonds joinings.
A cellulase system consists of three major components according to their differences in catalytic functions: Endoglucanases (endo-1,4-β-D-glucanases or 1,4-β-D-glucan glucanohydrolase; EC 3.2.1.4, C1, EG and CEN derived from fungi and bacteria individually), cellobiohydrolases (exo-1,4-β-D-glucanases or 1,4-β-D-glucan cellobilhydrolase; EC 3.2.1.91; Cx, CBH and Cex derived from fungi and bacteria individually), and β-glucosidases (1,4-β-D-glucosidase; βG; BGL, EC 3.2.1.21). The EG acts on insoluble cellulose surfaces, breaks internal bonds to disrupt crystalline structures of the cellulose and expose individual cellulose polysaccharide chains, and makes hydration of the cellulose chains easily. The CBH cleaves 2-4 units from ends of the exposed chains produced by the EG, resulting in tetrasaccharides or disaccharide such as cellobiose. There are two main types of the CBH a first type working processively from a reducing end, and a second type working processively from a non-reducing end of the cellulose. The βG hydrolyses CBH products into individual monosaccharides. Through synergistic actions of the above enzyme system, the cellulose can be efficiently hydrolyzed to glucose.
The cellulase's enzymatic reaction is different from general enzymatic catalytic reactions, lying in that the cellulase is a multicomponent enzyme system, and the structures of its substrates are extremely complex. Because of the insolubility of the substrates, an ES formation process in general enzymatic reaction could not be accomplished. Instead, the adsorption prosperity of the cellulase makes it adhering to the cellulose substrate. and the celluase catalyzes the celluose into the glucose under the synergistic actions of several components of the cellulase.
In 1950, Reese et. al proposed a C1-Cx hypothesis, which stated that a different enzyme synergistic effect for hydrolyzing cellulose completely into the glucose. A synergistic effect is generally considered that a endoglucanase (C1 enzyme) amorphous region first attack ingcellulose, the formation of Cx required a new free end, and then by the Cx enzyme from the reducing end of polysaccharide chains or non reducing end cutting fiber into two sugar units, and finally by the beta glucanase will hydrolyze them into two glucose. However, the sequential collaborative effect of the cellulase is not absolute. Found in subsequent research, C1-Cx and beta glucanase must all be present to hydrolyze natural cellulose. If t C1 enzyme was added first to act on crystalline cellulose, then replaced with Cx enzyme, the crystalline cellulose could not be hydrolyzed.
Breeding is a foundation of cellulase production domestic and foreign experts have done a lot of research on productions of the cellulase products with high quality. For example, Wang Jialin (1996), has introduced Trichoderma 10, Trichoderma viride Sn-91014, Trichoderma koningii NT-15, Aspergillus niger XX-15A. From them, Wang obtained high yield strain NT15-H, NT15-H1, XT-15H, XT-15H1 by using ultraviolet radiation, specific electromagnetic wave radiation, linear accelerator, and NTG mutagenesis method of physics and chemistry. The Trichoderma NT-15H solid culture activity by the Ministry of light industry food quality supervision and testing center of NanJing Railway Station detection showed that filter paper enzyme activity was 3670 u/g, C1-, 24460 u/g enzyme activity, Cx-enzyme activity of 1800 u/g, has reached an international superior level. The strains maintain stable qualities in industrial production. Zhang Linghua (1998) selected Trichoderma koningii W-925, J-931, which were induced mutations by 2% diethyl sulfate and ultraviolet (15W, 30 cm, 2 min) inductions, to produce high enzyme activity of Wu-932 strain. said strain has CMC saccharification enzyme activity of 2975 and 531, which equaled to 100% and 81% increase from that of strain W-925. Wang Chengshu from Department of chemical feed additive technology service center (1997) mutated Trichoderma reesei A3 by UV and NTG treatments. The spores were inoculated in a fiber double plate for 5-8 days at 30° C., then 7-10 days at 15° C. Single colonies having transparent circles and large diameters were selected and transferred to flasks for a solid-state fermentation screening. Trichoderma reesei strain 91-3 with very high cellulase activity was obtained.
There are two main kinds of production technologies of the cellulase, namely solid fermentation and liquid fermentation, the process is as follows:
Various factors could affect the yield of enzyme and its activity, including microbe species, culture temperature, pH, moisture, substrate, and cultivation time. These factors are not independent but interrelated. Adopting uniform design method C112 (1210) and Trichoderma viride (T.ViriclePers.expr) as strain, Zhang Zhongliang (1997) investigated the effect of five major factors on the production of the cellulase and its enzymatic activity. Zhang found that the culture conditions of 40% matrix crude fiber content, at initial pH7.5, with 4 times of water, at a temperature of 26-31° C. for 45 h would provide the maximum yield of enzyme of 26 mg/g and CMC enzyme activity of 20 mg/g·h−1. Wang Chenghua (1997) has also studied Trichoderma reesei 91-3 enzyme producing condition. His results showed that the strain 91-3 fermented best in a medium composed of a mixture of 7:3 of straw powder and wheat bran, with 4% ammonium sulfate, 0.4% potassium dihydrogen phosphate, and 0.1% magnesium , at 28-32° C. or preferably at 30° C. for 96 h. Zhang Linghua (1998) studied optimal fermentation conditions of mutated Wu-932 cellulase producing strains of Trichoderma koningii W-925 as starting strain,. The results showed that, a ratio of 1:2 of wheat bran and rice straw powder medium, an inoculation amount of 5%, an average length of 3-5 mm of straws, an initial pH4-5, a temperature of 28-35° C., and a fermentation time of 72 h were the optimal fermentation conditions.
A preparation method of the cellulase was mentioned in CN201010040047 by Zhejiang University, which contains following steps: Ricem (Trichderma reesei) were inoculated into a fermentation medium with a fed-batch culture pattern, wherein the fermentation medium was fed to the fermentation system when the pH of the fermented broth is higher than 4.8 , and the feeding was stopped when the pH was lower than 4.5; the total fermentation time was extended to 192-240 hours. Moreover, said invention introduced a combination of an insoluble and a soluble carbon sources to induce an expression of cellulase genes. The balance in microbe growth and metabolite production is maintained through fed batch culture, which solved problems when cellulose or soluble sugar was used as inducers for the cellulase production, effectively improving the level of cellulase fermentation.
A preparation method of a low-temperature neutral cellulase was invented as CN2013102687288. Trichoderma reesei was used to produce the cellulase through of a serial of steps: the seed was activated, UV mutagenesised, cultured at low temperature and purified; then, a resulting seed solution was inoculated to a producing-medium, cultured for 96-144 hours at 10-15° C. for the cellulase production. The invention also provides a method of compounding the low-temperature neutral cellulase. The beneficial effects of the invention were: simple operation, short fermentation cycle, reduction of the temperature of the application of enzyme, leading to reduction of the power consumption of industrial applications, which make it more environmentally friendly, and has wide application prospect.